Several transfer RNA molecules will be purified to homogeneity and studied by high-resolution NMR spectroscopy. Individual base pair resonances from the hydrogen-bonded ring NH protons will be resolved in the low-field NMR spectrum and will be assigned to specific secondary and tertiary base pairs by chemical, biochemical and spectroscopic means. Temperature-dependent studies will reveal the thermal unfolding sequence of these tRNA species in a variety of buffer, magnesium and ionic strength conditions. Saturation recovery FTNMR (using Redfield 214 observation pulses) will be used under conditions where recovery is exchange-dominated to determine the dynamic helix-coil breathing rates of selected strategic secondary and tertiary base pairs individually. Temperature-dependent helix-coil dynamics will be used to determine activation energies for unfolding specific tertiary base pairs. Four synthetases will be isolated and studied in complexes with each of their isoacceptor tRNA substrates by high-resolution NMR. Attempts will be made to crystallize these aminoacyl-tRNA synthetases, pure tRNAs and synthetase-tRNA complexes. If well-ordered single crystals are obtained they will be used for structure determination by x-ray diffraction. The structure of aminoacylated tRNA will be studied in solution by NMR and, if successfully crystallized, by x-ray methods. The aminoacyl-tRNA:EFTu complex will be studied in solution by NMR and attempts will be made to crystallize this complex.